| Abstract |
Opisthorchiasis caused by Opisthorchis viverrini (Ov) is an important parasitic foodborne disease in many parts of Southeast Asia including Thailand, Lao PDR, Vietnam, and Cambodia. Chronic infection is associated with several hepatobiliary diseases, especially gallbladder and bile duct inflammation (cholecystitis and cholangitis), periductal fibrosis, and cholangiocarcinoma, the fatal bile duct cancer. The mechanism by which this carcinogenic liver fluke induce chronic infection is currently unclear. This study was aimed to characterize and functional analysis of calpain, a tegumental protein of Ov. We evaluated the functional activity of calpain via hydrolysis of fluorogenic peptides and biological substrates. Enzymatic activity of native calpain in the crude somatic extracts was tested against calpain-specific fluorogenic peptide substrate N-succinyl-Leu-Leu- Val-Try-7-amino-4-methylcoumarin (AMC) calpain in assay buffer (100mM sodium acetate, 100 mM NaCl, 5 mM CaCl2 and 1 mM dithiothreitol, pH 2,3,4.5,5.5 or 100mM Tris-HCl, 100 mM NaCl, 5 mM CaCl2 and 1 mM dithiothreitol, pH 6.5,7.5,8.5,9.5). For inhibitory assay, somatic worm extracts protein was pre-incubated at 25°C for an hour with assay buffer at pH 4.5 with each protease inhibitors (100 μM E64, 5 mM EDTA, and 1 mM protease inhibitor cocktail), before adding the substrate. The enzymatic activity assay was measured by monitoring the release of fluorescence (AMC) upon hydrolysis of the substrate at 37°C for 60 min with 10 min interval using a fluorometer at 360 nm excitation and 460 nm emission. The result showed the maximum activity of calpain was at pH 4.5, which is different from other protease of Ov that has been studied to date. The proteolytic activity was inhibited by EDTA, protease inhibitor, and E64. In addition, it digested mouse IgG, human haemoglobin, and human plasma fibronectin, which suggested its function in immune evasion and nutrient acquisition. |
Publication
Journal Publication
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